Receptor-mediated endocytosis is an important step in many biological processes including regulation of surface receptors, uptake of iron and cholesterol, entry of some viruses and toxins, clearance of proteases form extracellular fluids, and antigen presentation. Molecules or particles brought into the cell by endocytosis can reach various cellular destinations. Many receptors recycle back to the cell surface as their ligands are degraded. Viruses and toxins enter the cytosol. A key step in many of the sorting events is exposure to a low pH in an endosome. There are several distinct types of endosomes. These include early peripheral small visicles, a sorting endosome (CURL), and two post-segregation endosomes: one returning to the cell surface and one leading to lysosomal degradation. Recent work from this laboratory has shown that the different types of endosomes maintain different pH vlaues. It is proposed that changes in acidity in part determine the functional properties of different types of endosomes. Using fluorescein labeled molecules as a pH proble, the pH of each of the specific types of endosomes will be measured. Most important will be the pH in the sorting endosome, which has never been measured apart from other types of endosomes. Mechanisms of pH regulation will also be studied in living cells by measuring the pH of specific compartments under altered contitions (temperature, drugs, ionic environment, etc). The origins and fates of these various endosomes and their interrelationship is not known. The fusions and separations of these endosomal compartments with each other will be studied using fluorescence microscopy, biochemical techniques, and electron microscopy. This should establish the lifetime of the sorting endosome and indicate whether the recycling endosomes can serve as sorting organelles. The molecular signals for carrying receptor proteins on the recycling pathway or to lysosomes will be investigated using transfectants of normal or altered EGF and transferrin receptors.